In this study, isolated spermatogonial stem cells (SSCs) and Sertoli cells using enzymatic digestion from patients with maturation arrest of spermatogenesis were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum in three different groups: (1) SSCs cultured without Sertoli cells (2) SSCs co‐cultured with Sertoli cells (as control group), (3) SSCs co‐cultured with Sertoli cells and adding different concentrations of basic fibroblast growth factor (0.1, 1, 10 ng ml⁻¹) and human leukaemia inhibitory factor (1000, 1200, 1500 unit ml⁻¹) as experimental groups. The assessment of colonies every 10 days during 5‐week cultures showed that in the first group, the average number and diameter of the colonies were significantly lower than in the other groups (P < 0.05). The largest number of colonies was observed in control condition (32.29 ± 9.15) in day 30. The largest diameter of colonies was formed in combination dosages of 1 ng ml⁻¹ basic fibroblast growth factor (bFGF) + 1500 unit ml⁻¹ leukaemia inhibitory factor (LIF) (302.93 ± 37.68) and 10 ng ml⁻¹ bFGF and 1200 unit ml⁻¹ LIF (262.87 ± 35.54) in day 30 respectively. Isolated SSCs were positive for spermatogonial cell markers such as Oct4, Stra8, Piwil2 and Vasa but negative for Nanog. Transplantation technique indicated that hSSCs have good efficiency for colonisation of mouse seminiferous tubules after proliferation in culture system.